This project investigates peripheral neural mechanisms that underlie the development of mechanical hyperalgesia; a prominent clinical feature associated with persistent muscle pain conditions. We have previously shown that peripherally localized NMDA receptor (NMDAR) and metabotropic glutamate receptor 5 (mGluR5) are important components in evoking acute muscle nociception as well as mechanical hyperalgesia. Several members of the transient receptor potential (TRP) family, particularly TRPV1 and TRPA1, also play an essential role in the development of mechanical hypersensitivity under various pain conditions. Since activation of peripheral glutamate receptors invokes various intracellular signaling cascades leading to nociceptor sensitization, and both TRPV1 and TRPA1 are suggested to function as 'inflammatory signal integrators', we propose that NMDAR/mGluR5 and TRPV1/TRPA1 functionally interact and that activation of NMDAR/mGluR5 leads to TRPV1/TRPA1-dependent mechanical hyperalgesia via multiple intracellular signaling pathways. Aim1 evaluates functional interactions between NMDAR/mGluR5 and TRPV1/TRPA1 with behavioral pharmacology and in vivo RNAi studies, and provides the morphological and biochemical bases for the interactions between the two receptor systems in trigeminal ganglia (TG). Experiments proposed under Aim2 investigate specific intracellular signaling pathways underlying NMDAR/mGluR5 and TRPV1 interactions, and Aim3 examines intracellular signaling mechanisms unique for NMDAR/mGluR5 and TRPA1 interactions. The integrated studies proposed here will provide comprehensive information on novel mechanisms of peripherally mediated mechanical hyperalgesia, and have immediate translational implications in a relatively understudied area of clinical muscle pain conditions, such as temporomandibular disorders. PUBLIC HEALTH RELEVANCE: This project examines cellular and molecular mechaisms that link two receptor-channel systems that have been independently implicated in muscle pain and hyperalgesia. Peripherally localized glutamate receptors such as NMDA and type I metabotropic glutamate receptors are being increasingly recognized as critical components in mediating the development of pathological pain conditions. We have convincing preliminary evidence that these glutamate receptors functionally interact with TRPV1 and TRPA1, members of the transient receptor potential family, and that interactions between the two types of receptor systems are important elements for mechanical hyperalgesia arising from craniofacial msucle tissue. Therefore, outcomes of this project can offer important new insights in the development of pathologic muscle pain conditions and mechanism-based treatment strategies that can be directed at the peripheral receptor systems to ameliorate persistent orofacial muscle pain conditions.